The Role of Livin, An Inhibitor of Apoptosis Protein, in Thrombopoiesis.

The exact mechanism of platelet production is poorly understood. A relationship between activation of the apoptotic cell machinery and the formation of pro-platelets has been established. In this study we show that Livin plays a major role in this process. Livin is a member of the Inhibitor of Apoptosis Protein (IAP) family, a novel family of intracellular anti-apoptotic proteins that act by binding and inhibiting caspases. We found that Livin mRNA and protein are expressed in platelets and Livin was also clearly detected by immunohistochemistry staining in normal mature bone marrow megakaryocytes (MK). Overexpression of Livin was also demonstrated in MK of patients with various hematological diseases such as ITP, MDS, Hodgkin’s disease, ET and PV. An in vitro model was established to evaluate the potential role of Livin in thrombopoiesis. The human erythroleukemic cell line, LAMA-84, was induced by a phorbol ester (PMA) to differentiate to MK. Differentiation was characterized by upregulation of the MK marker CD41 from 6% to >60% and downregulation of the erythroid cell marker CD71 from 97% to 70%. Increased cell size, ploidy, and DNA synthesis, all markers of MK differentiation, were detected by flow cytometry. Upon differentiation induced by PMA, LAMA-84 cells formed pro-platelets and produced functional platelets capable of aggregation. This thrombopoiesis was accompanied by Livin overexpression. Moreover, overexpression of Livin by transfection decreased the percentage of the undifferentiated CD71+ cells to 53 % compared to 73 % in control cells (p= 0.007) and increased the production of platelets by two folds compared to control cells. Overexpression of Livin reduced caspase 3 activity and inhibited MK apoptosis. Our results show that anti-apoptotic Livin plays a role in thrombopoiesis possibly by protecting the maturing MK from apoptosis, thus affording more time for efficient platelet production by longer living pro-platelets. We are currently using this new cell line model to further characterize the molecular relationship between apoptosis and thrombopoiesis.